Duplication of the DNA genome is essential to the life of all cells. To date, no other lab has succeeded in reconstituting the enzymology of the eukaryotic replication fork. This is in part due to the numerous proteins required and the difficulty in obtaining them. Unlike bacteria, eukaryotes use different multiprotein DNA polymerases to duplicate the two (leading and lagging) strands of DNA. The eukaryotic helicase is composed of 11 distinct proteins, while bacteria simply use a homohexamer. In addition, there are numerous proteins that function at eukaryotic replication forks that have no homologue in bacteria. We have recently succeeded in reconstituting a eukaryotic (Saccharomyces cerevisiae) replisome from pure proteins. The reconstituted replisome utilizes 31 distinct proteins to duplicate both leading and lagging strands of one DNA molecule. The reconstituted process requires the 11-subunit helicase among several other factors, and recapitulates the asymmetric utilization of the distinctive polymerases in eukaryotic cells. Thus only DNA polymerase epsilon (Pol e) functions on the leading strand and DNA polymerase delta (Pol d) functions on the lagging strand. These Pols are prevented from working on the wrong strand in the reconstituted system. Questions to be addressed in this proposal include: What mechanism prevents Pol d function on the leading strand, and what prevents Pol e from working on the lagging strand? The reconstituted fork travels 5-10 fold slower than forks inside cells, but we are missing many proteins that are known to travel with forks. We will purify these other factors and examine their function in moving replisomes. Do one or more of them speed the fork? Do they affect lagging strand priming frequency, replisome processivity, or replisome stability? We find that many replisome proteins associate to form a large assembly, even in the absence of DNA. The smallest of these is a 15 subunit helicase-Pol e complex. We have solved a 3D EM structure of this leading strand replisome complex in collaboration with Dr. Huilin Li (Brookhaven National Labs), and it shows several unexpected features. We have reconstituted larger complexes as well. We propose to solve the 3D structures of these complexes using cryoEM, as well as collaborate with Dr. Brian Chait (Rockefeller University) to identify protein connectivity t high resolution within the complexes by new innovative cross-linking/mass spectrometry techniques. In overview, the studies proposed here will provide a deep understanding of the workings of the eukaryotic DNA replication machinery. This process is intimately involved in DNA repair, mutagenesis, and human disease. Replication is also crucial for a positive response to many anticancer drugs that are currently in use. Hence detailed knowledge of this central and vital process to cellular life will provide important information that will be useful t prevention and cure of human disease, and maintenance of a healthy state.